Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer.
Genet Mol Res
; 6(2): 250-5, 2007 May 07.
Article
em En
| MEDLINE
| ID: mdl-17573654
ABSTRACT
Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.
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Base de dados:
MEDLINE
Assunto principal:
Análise de Sequência de DNA
/
Primers do DNA
/
Fluoresceína
/
DNA Polimerase I
/
Escherichia coli
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article